Approach 1: Human Papilloma Virus (HPV) pseudovirions, is a technology developed by my collaborators, Drs Buck, Schiller, and Lowy, at the NCI, delivers DNA to keratinocytes in the vaginal mucosal epithelium. So with this approach, it may be possible to elicit effector T-cells in the female genital tract. However, a limitation could be that,as the expression of SIV/HIV genes by this vaccine modality wanes with time,repeated immunizations may be required to maintain protective levels of effector T-cells in the vagina. Rhesus macaques have been immunized intravaginally with HPV 16 and HPV 45 constructs containing the SIV gag gene and immune response evaluated at mucosal and systemic sites. RMs were given systemic progesterone treatment, intramuscular Depo-Provera 30mg/kg, to thin the vaginal epithelium. Animals were treated with either 10% N9 gel or 12% N9 foam. A group of animals were also treated with a cytobrush to lightly abrade the vaginal wall. If the HPV vaccination induces and sustains polyfunctional memory responses in the vagina, then additional HPV constructs expressing early SIV genes (Retanef) and envelope will be generated. The SIV envelope DNA sequence will be derived from the sequence of SIV genotype transmitted to RM following vaginal challenge exposure. The envelope will be engineered such that it will be secreted. The overall goals of the second phase are to evaluate the impact of T-cell responses to SIV genes induced at the mucosal and systemic sites in the protection against a homologous SIV vaginal challenge. In addition, the role of binding and/or neutralizing antibodies to the envelope, particularly if present in vaginal secretions, will be evaluated. Approach 2: Induce effector T-cells in the gastrointestinal tract and will use heat shock gp96-Igbased SIV recombinant vaccines. This is part of a collaborative effort with Dr Podack from the University of Miami and includes the preparation of cellular SIV-gp96-vaccines secreted by 293 cells. Optimal dose-finding for SIV-gp96 vaccines in non-human primates was determined by specific CD8 responses. These cell-based gp96-Ig vaccines, by prolonged in vivo secretion of gp96-Ig-peptide, should imitate viral replication and provide immune stimuli comparable to attenuated viruses. In model systems in mice, Dr. Podack has shown that gp96-Ig transfected, antigen expressing tumor cells secrete gp96-Ig in vivo and stimulate the innate DC and NK as well as adaptive, cognate cellular CD8 CTL immune response and generate specific CD8 memory independent of CD4 help and in the absence of lymph nodes. Both systemic and strong mucosal immunity in intraepithelial, lamina propria, and Peyers patch CD8 CTL is generated by gp96-Ig vaccines. Because of their unique properties, we now plan to evaluate the gp96-vaccines in nonhuman primate models for SIV for their immunogenicity at mucosal and systemic sites (R21). In addition, we will examine the protective power of SIV-gp96-vaccines against subsequent viral challenge (R33). To maximize the chances of success, the team in Miami, Dr. Podack and Dr. Pahwa have entered into collaboration with my lab bringing together basic immunologists with expertise in human and non human primate HIV/SIV pathogenesis. We have constructed gp96-Ig vaccines encoding Gag, Env, and a chimeric Retanef protein and demonstrated the secretion of these proteins in the supernatant of 293 cells (see picture above on the left). We have completed the R21 phase and demonstrated that 3 intraperitoneal immunizations with 50 mg of secreted gp96-Ig protein induce SIV-specific functional responses in intra epithelium lymphocytes of the rectal mucosa of NHP (see picture on left). The aims of the R33 part of this grant are to generate mucosal and systemic SIVimmunity with secreted gp96-Ig vaccines and expose macaques to SIV by the intrarectal route to test the relative efficacy of these vaccines. Approach 3: Vaccine approach is based on an attenuated HTLV-II vector. HTLV-II infects dendritic cells, persists in the host T-cells, and is expressed at low level in the gut associated lymphoid tissues (GALT) and lymph nodes. The goal is to generate HTLV-II vectored-SIV vaccines that persist in the host and investigate whether the elicitation of low level, durable, effector T-cells in the gut, and central memory cells in lymph nodes will protect from SIV acquisition and dissemination to distal sites. These approaches in NHP should inform on the quality, quantity, and location of CD4+ and CD8+ memory cells necessary to prevent early seeding of SIV. Approach 4: This is not a new approach, but we are still investigating the relative merit of the ALVAC as a deliver platform for an HIV vaccine. ALVAC is an attenuated canarypox derived vector that cannot replicate productively in mammalian cells. ALVAC HIV-1 vaccine is a candidate HIV-1 vaccine, which is now in Phase III clinical trials and the results of this trail will become available at the end of September 2009. We have designed a study to improve the immunogenicity of an ALVAC SIV vaccine with the goal to test in parallel its efficacy against the high dose or the low dose repeated challenge exposure. In the past, we have tested an ALVAC-SIV vaccine candidate for HIV in an infant macaque model to assess whether this vaccine platform could reduce SIV transmission through breast-feeding. Infant macaques were given multiple immunizations during the first 3 weeks of life with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins Gag, Pol, and Env (ALVAC-SIV or modified vaccinia virus Ankara [MVA]-SIV). After repeated daily oral exposure to low doses of virulent SIVmac251 significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. Monkeys not infected after oral challenge in infancy were rechallenged at 16 months of age or older by repeated weekly oral SIV exposure;unimmunized animals were infected after fewer SIV exposures than were animals vaccinated with ALVAC-SIV. When infected, ALVAC-SIV -vaccinated animals also had reduced viremia compared with unimmunized animals. These results suggested that immunization of human infants with poxvirus-based HIV vaccine candidates may offer protection against early and late HIV infection and were in contrast with the poor efficacy of these vaccines in adult macaques. It is unclear whether the age or the mode of challenge is responsible for the different degree of protection observed in these two studies. Thus, we are further investigating this approach for the following reasons: 1) ALVAC is an avian vector;therefore, pre-existing immunity is not a factor that could enhance HIV infectivity. This is particularly important in light of the recent results with the Ad5-based HIV vaccine trial (STEP trial) wherein a five fold increase in HIV transmission was observed in individuals (not circumcised)that had high levels of pre-existing immunity to the vector. 2)ALVAC-HIV alone may not be sufficiently immunogenic or protective.3)We will use this study to test in parallel the degree of protection from a mucosal challenge exposure to high and repeated low doses of SIVmac251. Hopefully, the results of this study compared to the results of the Thailand Trial, will inform on the relevance of the two challenge models in predicting the efficacy of vaccines in humans. We also plan to characterize and compare the viral genotypes that are transmitted early in nave and vaccinated RMs by cloning and sequencing the vi [summary truncated at 7800 characters]